ABSTRACT Membrane Protein Folding and Assembly (GM74637) Many human diseases, such as cystic fibrosis, result from misfolding of membrane proteins (MPs) during their synthesis and targeting. It is therefore important to understand the principles and mechanism of MP folding and assembly. A largely unexplored part of the problem is to understand folding in the context of the cellular milieu. Toward that goal, we are studying the targeting, secretion, and insertion of membrane proteins along the so-called SecA post- translational pathway of living Escherichia coli. We showed earlier that the SecA motor ATPase, a significant drug target, can insert single-span membrane proteins (S-SMPs) across the E. coli inner membrane. This simplified in vivo model system eliminates the many unanswered questions about the folding of multi-span MPs along the signal recognition particle (SRP) pathway, because we gain direct access to the translocon-bilayer partitioning process. We have engineered two different chimeric protein families for probing systematically S- SMP stability using TM segments of the form GGPG-H-GPGG (used in an earlier study to determine a biological hydrophobicity scale using a cell-free eukaryotic system). To determine stabilities, we have developed methods for cleaving TM segments in vivo via native intramembrane proteases. We have discovered that many S-SMPs are stable across the membrane only because their periplasmic & cytoplasmic domains cannot cross the membrane. We have also discovered that translocon-to-membrane transfer energetics are not equal to membrane-to-cytoplasm transfer energetics and that stability depends upon growth temperature. Little is known about SecA function at the atomic level despite hundreds of papers on the subject. Calling upon our lab?s expertise in lipid-protein interactions, we have begun electron cryomicroscopic (cryo-EM) studies of the structure of SecA bound to lipid nanodiscs. Our specific aims for the proposed research are the following: (1) Determine an in vivo membrane-to-cytoplasm hydrophobicity scale. (2) Determine an in vivo translocon-to- membrane hydrophobicity scale. (3) Determine why the scales depend upon temperature, which we hypothesize is due to temperature-dependent inner membrane lipid composition. (4) Develop in our laboratory cryo-EM tools for structural determinations of SecA in solution and when bound to nanodiscs with the distant goal of describing each step of the secretion process at the atomic level.